The free energy of hybridization of complementary hybridizing oligomers such as nucleic acids and nucleic acid analogs can be directed to effect controlled and reversible changes in the configuration of molecular structures, and so can be used to control a molecular switch, or can serve as fuel to drive the motion of a nanomachine. This invention pertains to methods in which oligomer hybridization/displacement reactions operate a reversible molecular switch, or fuel a nanomachine. The methods of the invention comprise controlling the number and type of hybridizing subunits, e.g., nucleotides, in single-stranded xe2x80x9ctoeholdxe2x80x9d regions that extend from double-stranded oligomer complexes attached to the molecular switches or nanomachines. The single-stranded toehold regions enhance the rate with which single-stranded oligomers hybridize to and displace one of the strands of such double-stranded complexes to reversibly alter the configuration of the molecular switches or nanomachines. The invention further includes molecular switches and nanomachines comprising molecular structures which reversibly assume alternate configurations at molecular reaction rates that are controlled by hybridization reactions involving said oligomers comprising single-stranded xe2x80x9ctoeholdxe2x80x9d regions.
It has been shown that a single-stranded region of a nucleic acid extending from the end of a double-stranded (duplex) complex formed by hybridization of two strands of unequal length provides a nucleation site, or xe2x80x9ctoehold,xe2x80x9d for hybridization of a third nucleic acid strand complementary to the longer strand. A toehold-mediated hybridization/displacement reaction is initiated when a portion of the third strand hybridizes to the single-stranded toehold sequence, and proceeds with the remaining portion of the third strand subsequently hybridizing to the longer strand while displacing the shorter strand. The rate of strand displacement by such a toehold-mediated hybridization/displacement reaction is considerably greater than the rate of the strand displacement reaction when there is no toehold region [1-3]. Methods utilizing toehold-mediated hybridization/displacement reactions have been developed for detecting nucleic acids having specific nucleotide sequences [2, 3], and for ligating linker oligonucleotides to nucleic acids having specific nucleotide sequences to facilitate detection, affinity chromatography, and cloning of the nucleic acids [4, 5].
There have been several studies of the effects of varying biochemical and physical parameters of the toehold-mediated hybridization/displacement on the rate of the reaction. The overall rate of a toehold-mediated strand-displacement reaction is limited by the rate of association of the xe2x80x9cincomingxe2x80x9d displacing strand with the toehold region; the displacement of the shorter strand is not rate-limiting ([2], p. 1635; and [6], p. 4210). Once the displacing strand associates with the toehold region and begins displacing the shorter strand, the shorter strand is displaced via double-strand branch migration with a rate of approximately 12 xcexcsec per nucleotide (determined in 0.3M NaCl, at 65xc2x0 C.; see [2], p. 1635, and [7], p. 1911). The overall rate of hybridization/displacement reaction is increased by (1) increasing the is temperature, (2) increasing the concentration of the displacing DNA strand, and (3) adding volume-excluding polymers, presumably by increasing the rate of association of the incoming strand with the toehold region ([2], p. 1635; and [8], p. 20). The hybridization/displacement reaction rate is also increased by (4) modifying the displacing DNA strands so that they contain 5-bromodeoxycytidine (BrdC) or 5-methyl-deoxycytidine (MedC) nucleotides, which increase the affinity with which the displacing strand hybridizes to its complementary DNA strand in the duplex ([5], p. 2251; and [6], p. 4207). The rate of the hybridization/displacement reaction can also be increased by (5) increasing the G+C content of the toehold region ([6], p. 4207), (6) adding Rec A protein to the reaction mixture ([8], p. 25), and (7) using a displacing strand which is double-stranded at its terminal portion adjacent to the single-stranded region that binds the toehold sequence ([6], p. 4210). The effects of the lengths of the displacing strand and the displaced strand on the overall reaction rate are not well understood, and appear to be dependent on the nucleotide sequence of the toehold region, and possibly on the nucleotide sequence of the duplex region adjacent to the toehold region (compare [2], p. 1635, and [6], p. 4211). The effects of mismatches between the bases of the incoming, displacing strand and those of the longer, toehold-linked strand on the rate of the strand displacement are also unclear, and appear to depend strongly on the temperature of the reaction. For example, one study reports that a single mismatch blocks branch migration at 55xc2x0 C. ([4], p. 8680), whereas another study reports that a cluster of 5 mismatches among 7 bases does not reduce the efficiency of strand displacement at 65xc2x0 C. ([8], pp. 23-24). Interestingly, the latter study also reports that 27% base mismatch over 85 nucleotides blocks strand displacement at 65xc2x0 C., but not at 55xc2x0 C. ([8], pp. 23-24). The latter result indicates that displacing strands can be synthesized having a selected number of mismatches so that displacement does not occur unless the temperature is at or above a selected value.
It has been shown that a toehold-mediated hybridization/displacement proceeds approximately 3 times more rapidly with the 4-nucleotide toehold sequence GGCC- than it does with the 3-nucleotide toehold CCG-([6], p. 4211); however, the precise relationship between the length of the toehold region and the rate of the toehold-mediated hybridization/displacement reaction has not been described prior to the present invention.
Molecular nanotechnology uses molecular engineering and manufacturing capabilities, employing the capabilities of biotechnology in combination with other technologies such as proximate probe technology and supramolecular chemistry, to develop nanometer-scale machines and devices assembled from natural and nonnatural macromolecules and other chemical structures [9]. An example of such a molecular device is a controllable two-state molecular switch, which can be used to store information in the same manner as the counters of an abacus, or the electronically operated switches of a computer ([9], pp. 2012-2014).
DNA nanotechnology takes advantage of the self-organizing properties of DNA polymers, and uses DNA oligomers having selected nucleotide sequences as structural elements in the assembly of complex structures on a molecular scale [10-14]. The advantages of using DNA to construct nanodevices include
(1) double-stranded DNA molecules of 1-3 turns are relatively rigid structural elements, and the intermolecular interactions of DNA are relatively well-understood and predictable, so that DNA polymers can be designed which will self-assemble in a predictable manner;
(2) oligomers of DNA and its analogs having arbitrary subunit sequences can be produced readily using solid support synthesis;
(3) many different methods for chemically modifying DNA have been developed, e.g. to attach linking functions, catalytic or structural polypeptides, or detectable groups such as biotin and fluorescent labels, and to modify properties of the DNA such as resistance to cleavage by nucleases, hydrophobicity, flexibility, and duplex stability;
(4) DNA can be manipulated by an array of enzymes that include DNA restriction endonucleases, DNA ligase, kinases, and exonucleases; and
(5) the external surface of DNA polymers is rich in structural information, and segments of single- and double-stranded DNA can be recognized and bound by other nucleic acids and by DNA-binding sites of proteins, in DNA-binding reactions having a wide range of specificities and affinities ([10], p. 228).
It has been proposed that DNA structural transitions such as migration of a cruciform branch structure, or the B-Z transition, might be used to drive a nanomechanical device ([10], p. 245). Prior to the present invention, there was no suggestion to capture and use the free energy of the transition from single-stranded DNA to double-stranded DNA to alter the configuration of a molecular switch, or to fuel a molecular machine.
A single-stranded terminal portion of a first nucleic acid strand that extends from an end of a duplex complex formed by hybridization of the first strand to a second, shorter, complementary nucleic acid strand, provides a xe2x80x9ctoeholdxe2x80x9d nucleation site for hybridization for a third single-stranded nucleic acid that is also complementary to the first one, and greatly enhances the rate of the hybridization/displacement reaction in which the third nucleic acid displaces the second strand and forms a double-stranded complex with the first strand, relative to the rate when there is no toehold. The present invention pertains to methods in which this toehold-mediated process of enhancing the rate of strand displacement is employed to direct the free energy of nucleic acid hybridization to do useful work; for example, to effect changes in nucleic acid configurations, to produce molecular switches, to fuel molecular machines, to drive molecular reactions, and to produce catalytic, or cascading, reactions which respond to, and permit detection of, very small amounts of a particular nucleic acid. The methods of the present invention employ toehold-mediated strand-displacement to cyclically or reversibly alter the configuration of a molecular nanomachine comprising or attached to one or more xe2x80x9cmotorxe2x80x9d nucleic acid strands. A nanomachine operated by the reversible or cyclical binding of a fuel strand to and from a motor strand according the present invention can function as a molecular motor that periodically applies a discrete, significant force to a portion of a separate molecular device to which it is attached or with which it makes contact. Alternatively, such a nanomachine can operate as a reversibly controllable switch or signalling device; for example, binding of the fuel strand to the motor strand can put the switch in the xe2x80x9conxe2x80x9d position, and toehold-mediated displacement of the fuel strand by the removal strand can provide a means for returning the switch to the xe2x80x9coffxe2x80x9d position. One or more fluorophores can be attached to the nanomachine, to one or more motor strands connected thereto, or to the fuel strands, in such a manner that the change in physical configuration of the nanomachine and motor strands resulting from hybridization of a fuel strand to one or more motor strands results in a change in the fluorescence of a fluorophore, thereby providing a detectable signal indicative of the current configuration of the nanomachine.
Reaction kinetics measurements for the toehold-mediated process of enhancing the rate of nucleic acid strand displacement are disclosed herein which indicate the rates with which such molecular motors, switches, and reactions, could be driven.